Human vitronectin is a glycoprotein present in both the circulation and the extracellular matrix, where it interacts with a wide variety of other macromolecules and provides regulation of many physiological processes. Circulating vitronectin associates with the extracellular matrix of the vasculature, and its presence is important in pathological situations or in cancer metastasis and tissue remodeling. Since vascular cells do not synthesize vitronectin, the protein is incorporated into the matrix from a circulatory pool. The factors that control the binding interactions and determine activities of vitronectin in the circulation as well as in the matrix are important unsolved issues. We set forth the following as our working hypothesis regarding vitronectin complexes and their activities: complexes of vitronectin with biological targets (plasminogen activator inhibitor type 1 or the antithrombin-thrombin pair) associate into higher order structures. Vitronectin complexes are recognized by cell-surface receptors, with a preference for higher order complexes over free monomeric vitronectin. Vitronectin is a multi-domain protein with distributed binding sites to control the dynamic processes of coagulation versus fibrinolysis and binding versus release from the matrix. Our recent observation that PAI-1:vitronectin complexes associate into higher order structures lead us to propose that binding to relevant physiological targets can give a clustering of binding sites that is advantageous for binding to physiological surfaces, vitronectin by: 1. thoroughly evaluating of the mechanism by which the PAI-1:vitronectin complex forms, including an assessment of relative affinities of two putative PAI-1 binding sites; 2. determining whether other ligands which bind vitronectin in the circulation (notably the thrombin-antithrombin complex) give rise to higher order complexes; 3. observing the facility with which the biological complexes associate with the extracellular matrix and/or become endocytosed, and 4. continuing to use biochemical and recombinant methods to evaluate a domain organization and structural requirements for ligand binding. Our results will shed light on the activity and processing of complexes of vitronectin with target molecules and will provide a better understanding of how multiplicity in binding sites is used to advantage in this and other systems, including other matrix-associated proteins that are multifunctional and oligomeric in nature.